A phage-displayed cyclic peptide that interacts tightly with the immunodominant region of hepatitis B surface antigen

Abstract

The surface antigen (HBsAg) of hepatitis B virus (HBV) is highly conformational and generally evokes protective humoral immune response in human. A disulfide constrained random heptapeptide library displayed on the coat protein III of filamentous bacteriophage M13 was employed to select specific ligands that interact with HBsAg subtype ad. Fusion phages carrying the amino acid sequence ETGAKPH and other related sequences were isolated. The binding site of peptide ETGAKPH was located on the immunodominant region of HBsAg. An equilibrium binding assay in solution showed that the phage binds tightly to HBsAg with a relative dissociation constant (KDrel) of 2.9+/-0.9 nM. The phage bearing this peptide has the potential to be used as a diagnostic reagent and two assays for detecting HBsAg in blood samples are described.

Design of virus-based nanomaterials for medicine, biotechnology, and energy

“…For example, phages decorated with peptides isolated from phage display have been used in ELISAs for the detection of anthrax spores 383 and the surface antigen of HBV. 384 For an alternative to the presentation of peptides on viruses, antibodies can also be presented on the viral scaffold for multivalent detection, and the previous section discussed how phage display and genetic engineering could be used for the display of antibodies and fragments thereof. As another approach, it was recently demonstrated that functionalization of PVX-based nanoparticles with protein A fragments can be used to display whole antibody molecules by using protein A's property of binding to the Fc region of the heavy chain of IgGs.…”

A phage-displayed single chain variable fragment that interacts with hepatitis B core antigen: Library construction, selection and diagnosis

“…This suggests that C4 phage is most likely interacted with the immunodominant region of HBcAg. The negative control using an unrelated peptide with the sequence ETGAKPH that binds tightly to HBsAg (ad subtype) (Tan et al, 2005) did not inhibit the binding of the C4 phage to HBcAg.…”

“…ABTS was added and A 405 deter- mined. The nucleotide sequence of the insert was determined as described in Tan et al (2005) …”

“…The mixtures (100 l) were then added into wells coated with HBcAg, incubated at 4 • C for 2 h and washed six times with TBST. The bound phages were eluted with glycine-HCl (0.1 M, pH 2.2; 120 l) and the amount was determined by titration (Tan et al, 2005) and expressed as plaque forming unit (pfu).…”

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

“…Thus, it is considered to be antigenically most significant from a vaccine development point of view [5][6][7]. In addition, HBsAg are also needed to develop sensitive and specific immunoassays for in vitro diagnosis of anti-HBs antibodies as capture agents and reference markers [8,9]. Recombinant HBsAg has been successfully produced from the Pichia pastoris expression system and its advantages have been vastly reviewed in the literature [10][11][12].…”